Remineralisation of Calcified Tissue

ABSTRACT

A cosmetic and/or therapeutic treatment of tissue, such as tooth, is disclosed that effects, for instance, whitening and tissue re-building through mineralisation. Further, a method of performing iontophoresis utilizing an aqueous composition of a remineralising agent to achieve mineralisation is disclosed, as well as a kit for performing the mineralization or re-mineralisation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/668,684, filed May 10, 2010, which is a U.S. National PhaseApplication of PCT International Application No. PCT/GB2009/001009,filed Apr. 21, 2009, which claims priority of British Application GB0807224.1, filed Apr. 21, 2008. The entire content of these applicationsis incorporated herein by reference.

FIELD OF THE INVENTION

The invention concerns cosmetic and therapeutic treatment of tissue,such as tooth, to effect, for instance, whitening and tissue re-buildingthrough mineralisation.

BACKGROUND OF THE INVENTION

Iontophoresis is a non-invasive method of propelling high concentrationsof a charged substance, normally a medication or a bioactive agent,using a small electric charge applied to an iontophoretic chamber.

It is known to use iontophoresis in transdermal drug delivery. Also,iontophoresis is known to be used in conjunction with fluoridecontaining compounds to treat dentine hypersensitivity. Simone, J. L.,et al, Iontophoresis: An Alternative in the Treatment of IncipientCaries? Braz. Dent. J, 1995, 6(2), 123-129 describes, inter alia,treating dental lesions iontophoretically with sodium fluoride andclaimed to find good remineralisation due to the formation of calciumfluoride, though this was not validated.

CPP-ACP is a casein derived peptide, with added calcium and phosphate,specifically, casein phosphopeptide-amorphous calcium phosphate. CPP-ACPacts as a calcium and phosphate reservoir.

Conventionally, CPP-ACP is delivered to a tooth surface in severalvehicles, such as chewing gum, mouth wash, toothpaste and otherrestorative materials.

Thus, for example, International Patent Application No. WO 02/094204describes a composition for dental restoration including a dentalrestorative material and an effective amount of a caseinphosphopeptide-amorphous calcium phosphate (CPP-ACP) complex or caseinphosphopeptide-amorphous calcium fluoride phosphate (CPP-ACFP) complex.

When used herein, the term remineralisation is used to meanmineralisation of an area to which further material is being added,whether or not there was insufficient material at the area before thetreatment.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided a methodof remineralising tissue which comprises pre-conditioning, the tissue toremove protein and/lipids, and then applying to the tissue aremineralising agent whilst separately, sequentially or simultaneouslyapplying iontophoresis.

Preferably, the remineralising agent is a source of phosphate, calciumand water.

Preferably, the method comprises the remineralisation ofhypo-mineralised or demineralised tooth.

In one aspect, the method is a cosmetic treatment which is directed tolightening or whitening tooth.

The method may be directed to the prevention or treatment of tootherosion.

In another aspect the method may comprise the remineralisation of bone.

Preferably, the remineralising agent comprises caseinphosphopeptide-amorphous calcium phosphate (CPP-ACP).

The remineralising agent preferably contains fluoride. An example ofsuch a remineralising agent is casein phosphopeptide-amorphous calciumfluoride phosphate (CPP-ACFP).

Preferably, the remineralising agent includes one or moreremineralisation enhancers. Typically the remineralising, enhancers aresources of calcium and phosphate ions. Examples of remineralisationenhancers may include, but are not limited to Dicalcium phosphatedehydrate (DCPD), mineral brushite; Dicalcium phosphate anhydrous(DCPA), mineral monetite; Octacalcium phosphate (OCP); alpha-tricalciumphosphate (alpha-TCP); beta-tricalcium phosphate (beta-TCP); Amorphouscalcium phosphate (ACP); Calcium-deficient hydroxyapatite (CDHA);Hydroxyapatite (HA or OHAp); Fluorapatite (FA or FAp); Tetracalciumphosphate (TTCP or TetCP), mineral hilgenstockite). More preferably, theremineralisation enhancer is strontium.

The remineralising agent may include at least two remineralisationenhancers wherein one of the enhancers is a source of calcium ions andthe other is a source of phosphate ions. For example the remineralisingagent may include a source of calcium e.g. calcium hydroxide and asource of phosphate e.g. orthophosphoric acid. The ratio ofcalcium:phosphate in the remineralising agent may be between 1:1 and22:10. Preferably the ratio of calcium:phosphate is about 10:6 (i.e.1.67), which represents the ratio of calcium to phosphate ions incalcium hydroxyapatite. Alternatively, the ratio of calcium:phosphate inthe remineralising agent may be between 9:6 and 22:10. Alternativelystill, the ratio of calcium:phosphate in the remineralising agent may begreater than 1:1, but less than 3:2 (i.e. 1.0 up to 1.49).

The remineralising agents may thus be selected from the following: i)Ca:P ratio=1.67: e.g. Hydroxyapatite: Fluorapatite. Ca:P ratio=1.5-2.2(but not 1.67): e.g. Alpha-Tricalcium phosphate; Beta-Tricalciumphosphate; Amorphous calcium phosphate; Calcium deficientHydroxyapatite; Tetracalcium phosphate, mineral hilgenstockite. iii)Ca:P ratio=1−1.49: e.g., Dicalcium phosphate dehydrate, mineralbrushite; Dicalcium phosphate anhydrous, mineral monetite.

The remineralising agent may be prepared from its component parts by‘driving’ in calcium ions iontophoretically (in aqueous solution) andsubsequently changing the polarity of the set-up and ‘drive’ inphosphate ions (in aqueous solution) with a second sequence ofiontophoresis—the calcium and phosphate ions would thus ‘meet’ withinthe lesion during the second sequence of iontophoresis and precipitateout as a calcium phosphate mineral (or minerals). The hydroxyl ion ofthe generated apatite would come from the aqueous solution. Thewater-soluble calcium-containing agent might be, for example, calciumhydroxide, calcium chloride, or calcium nitrate; the water-solublephosphate-containing agent might be, for example, orthophosphoric acid(H₃PO₄) sodium (or potassium) hydrogen phosphate, sodium (or potassium)dihydrogen phosphate or magnesium phosphate. The calcium agentcontaining .solution may be separate from the phosphate agent containingsolution, or combined into one solution.

Thus s preferred method of the invention comprises the steps of: i)pre-conditioning the tissue to remove protein and/lipids, and ii)applying to the tissue a calcium-containing aqueous solution and/orphosphate-containing aqueous solution whilst separately, sequentially orsimultaneously applying iontophoresis. Optionally after sufficient timefor the ingress of a predetermined amount of calcium ions, determined(indirectly) by measurement of the amount of current discharged into thetooth, this first phase of remineralisation would be stopped and thepolarity of the iontophoresis electrode at that surface would be changedto negative; the remineralising agent would be changed to an aqueoussolution of orthophosphoric acid and the iontophoresis method re-appliedin order to cause the ingress of phosphate ions into the tooth. Thereversal of the previous iontophoresis polarity will cause thepreviously migrated calcium ions within the tooth to migrate towards thesurface as the phosphate ions are migrating into the tooth—thiscombination of calcium and phosphate ions, in aqueous solution, withinthe tooth will result in the deposition of orthophosphates within thetooth—i.e. remineralisation. This second phase of iontophoresis will bestopped when a pre-determined level of current has been discharged intothe tooth.

Thus a further preferred method of the invention comprises the steps ofi) pre-conditioning the tissue to remove protein and/lipids ii) applyingto the tissue a calcium-containing aqueous solution orphosphate-containing aqueous solution whilst separately, sequentially orsimultaneously applying iontophoresis, and iii) either (a) applying aphosphate-containing aqueous solution where in (ii) a calcium-containingaqueous solution was applied or (b) applying a calcium-containingaqueous solution where in (ii) a phosphate-containing aqueous solutionwas applied whilst separately, sequentially or simultaneously applyingiontophoresis.

Preferably, the pre-conditioning step is performed, with or without theapplication of iontophoresis, prior to application of the remineralisingagent/iontophoresis.

Preferably, the pre-conditioning step comprises treatment with an acid,more preferably, phosphoric acid.

Preferably, the pie-conditioning step comprises treatment with ahypochlorite.

A preferred method of the invention involves the treatment oralleviation of dental caries and/or dental fluorosis in a mammal.

A further preferred method of the present invention comprises theremineralising of hypo-mineralised or de-mineralised (carious) dentine.

The present invention also provides a remineralising agent for use iniontophoretic remineralising treatment of tissue which has been subjectto pre-conditioning to remove protein and or lipids, the remineralisingagent being a source of both phosphate and calcium.

Preferably, the remineralising agent comprises caseinphosphopeptide-amorphous calcium phosphate (CPP-ACP).

The present invention further provides a kit for use in iontophoreticremineralising treatment of tissue comprising a pre-conditioning agentand a remineralising agent.

Preferably, the pre-conditioning agent and the remineralising agent arepresent in the kit in a suitable form for application, for instance, aliquid or a gel form.

The kit may also provide an applicator for applying the or each agent tothe site of treatment

MORE DETAILED DESCRIPTION OF THE INVENTION

As indicated above, the present invention provides a method ofremineralising hypo-mineralised or de-mineralised tooth. However, themethod may be utilised in the remineralisation of other hypo-mineralisedor de-mineralised tissue, such as, bone.

A variety of remineralising agents may be used including a mixture ofremineralising agents. The remineralising agent may depend upon thetissue to be treated. However, preferably, the remineralising agent is aphosphate or calcium source, preferably a source of phosphate andcalcium. An especially preferred remineralising agent is caseinphosphopeptide-amorphous calcium phosphate (CPP-ACP). For use in theremineralisation of tooth, the remineralising agent may be a fluoridecontaining agent as hereinbefore described, such as caseinphosphopeptide-amorphous calcium fluoride phosphate (CPP-ACFP). Otherremineralising agents may comprise calcium phosphate compounds, such asfluoroapatite, monefite, brushite, amorphous calcium phosphate,hydroxyapatite, etc. Furthermore, it may be possible to incorporateadditional elements in the remineralising agent of the invention winchmay enhance the remineralisation effect, such as strontium.

It will be understood by the person skilled in the art that the termshypo-mineralised tissue and demineralised tissue are intended to includeany tissue that is deficient in its level of mineralization and includestissue, such as tooth, that is substantially or completelydemineralised, e.g. as a result of the dental caries process, thusincluding dental caries lesions, or a result of acid erosion, thusincluding ‘surface-softened’ enamel or dentine.

The iontophoresis may comprise the application of a voltage, e.g. afixed voltage, or a current, e.g. a fixed current. Alternatively, theiontophoresis may comprise the application of a mixture of voltage andcurrent, for example, the combination of voltage and current may beapplied in specific sequences so as to optimise remineralisation.

In addition, in the method of the invention a preconditioning step isalso included prior to application of the remineralisingagent/iontophoresis. The pre-conditioning step may vary but may, forexample, comprise the removal of proteins and/or lipids prior toapplication of the remineralising agent/iontophoresis. Although avariety of pre-conditioning steps may be used, preferably, thepreconditioning step comprises a variety of processes or a mixture ofprocesses.

Any suitable protein removing agent can be used in the preconditioningstep of the present invention. The agent is required to reduce theproteinaceous barrier formed over the surface to be treated, such as thepellicle over teeth or the exogenous protein within a caries lesion. Thepreconditioning step may optionally include the use of iontophoresis andthe various preconditioning agents, e.g. protein removing agents, may beused in a variety of combinations and/or sequences. Furthermore, any ofthe pre-conditioning agents may be propelled into a hypo-mineralised ordemineralised region, e.g. caries lesion, by iontophoresis to optimisethe disruption of the protein layer and then the polarity of theiontophoresis reversed in order to aid the removal the proteinaciousmaterial from the hypo-mineralised or demineralised tissue. Examples ofsuitable agents include bleach, detergent, chaotropic agents such asurea, high phosphate concentrations, cocktails of proteases (e.g.endopeptidases, proteinases and exopeptidases) and any other proteinsolubilising, disrupting or hydrolysing agent. Examples of suitablebleaches include sodium hypochlorite, and peroxide bleaches. In apreferred embodiment, the bleach is an alkaline bleach. In a furtherpreferred embodiment the alkaline bleach is sodium hypochlorite. Theprotein disrupting agent acts to solubilise and partially or whollyremove proteins from the surface of the tooth mineral, e.g. proteins ofthe pellicle on the tooth surface. However, preferably thepreconditioning step comprises treatment with an acid, such as anorganic, acid, e.g. acetic acid, an inorganic acid, e.g. phosphoric,acid, or a bleaching agent, e.g. hypochlorite, for example, sodiumhypochlorite.

The remineralising agent may be applied in a variety of forms, forexample, in the form of a gel or mousse. For use in the treatment oftooth other oral applications known per se may be used.

Pre-conditioning is preferably carried out not more than one minutebefore the application of the remineralising agent. More preferably, theremineralising agent is applied almost contemporaneously, i.e. withinseconds, of the preconditioning.

A preferred treatment sequence involves repeated conditioning followedby remineralising, particularly in a case where the remineralising agentincludes material, such as protein, which is removed in a subsequentconditioning step.

The present invention further provides a method of cosmetic treatment oftissue by application to the tissue of a remineralising agent whilstseparately, sequentially or simultaneously applying iontophoresis.

It will be further understood by the person skilled in the art that themethod of the invention may also be advantageous in the field oforthopaedics, for example, in the treatment of bone pathologies inmammals, i.e. human or animals, such as fractures and/or during surgery.

The present invention provides improved remineralisation of tissue.However, conventional methods of remineralisation of tooth generallycomprise remineralisation of the surface tissue, i.e. remineralisationof enamel. It is a particular advantage of the present invention thatthe method and/or use provide for remineralisation of dentine. Dentineis the term for a hard substance which is related to bone and forms thecore of the tooth in mammals and man. Dentine consists to the extent ofapproximately 30% of a cell-free organic base substance, in particularglycoproteins in which collagen fibres are incorporated. The inorganicconstituents are predominantly hydroxyapatite, fluoroapatite and smallamounts of carbonates, magnesium and trace elements.

The present invention further provides a kit for use in iontophoreticremineralising treatment of tissue comprising a pre-conditioning agentand a remineralising agent. The remineralising agent may comprise asource of calcium and phosphate ions such as defined herein.

Preferably, the pre-conditioning agent and the remineralising agent arepresent in the kit in a suitable form for application, for instance, aliquid or a gel form.

The kit may also provide an applicator for applying the, or each, agentto the site of treatment.

The EAER pre-treatment and iontophoresis remineralisation treatmentprocedure is implemented with the aid of a kit comprising several or allof the following: (1) the EAER remineralisation smart applicator pen;(2) battery pack and/or optional mains supply/recharger; (3) a set ofdisposable pre-treatment electrode pads which attach to the electrode ofthe EAER pen; (4) bottle of hypochlorite pre-treatment hydrogel, pasteor liquid; (5) a bottle of peroxide pre-treatment hydrogel, paste orliquid; (6) a set of disposable EAER remineralisation electrode padswhich attach to the electrode of the EAER pen; (7) one or more bottlesof hydrogel, paste or liquid containing the remineralisation agentsspecified above including: CPP-ACP, CPP-ACPF, etc; (8) all necessarywiring to complete the iontophoresis circuit, including a wrist-attachedor mouth-attached counter electrode; (9) full instructions. The gelscomplete the electrical path between the electrode pad and the tooth.Further optional add-on kits would supply dental trays, strips orholders or extension applicators.

The pre-treatment electrode pads (3) and remineralisation electrode pads(6) provide a disposable barrier between the EAER pen electrode and thegel for cross-infection control purposes, and also provide a support forthe hydrogel. Alternatively, the pads could be washable andsterilisable. They would preferably be composed of electricallyconductive material such as carbon-filled polymer or graphite felt, orhigh surface area silver/silver chloride electrodes. Alternatively, theymay be thin, non-conductive, open, porous sponge-like materials such assilicone or dried hydrogel which allow the applied hydrogel, paste orliquid to permeate throughout the material, providing an ionicallyconductive path to the underlying EAER pen electrode. in anotherembodiment the hydrogels may be applied directly to the EAER penelectrode without the use of an intervening, electrode pad (3) or (6).

To increase shelf-life, the pre-treatment gels or pastes (4) and (5)would preferably use an inorganic-based hydrogel or paste, such asinorganic gel formers tricalcium silicate, dicalcium silicate, andsodium silicate, or a non-reactive organic hydrogel such as polyvinylacetate, polyvinyl butyral, polyvinyl alcohols, hydroxymethyl cellulose,konjac, p-HEMA (polyhydroxyethylmethacrylate) andpolyoxypropylene-polyoxyethylene. Alternatively, the pre-treatment gelwould be prepared immediately before application by mixing the dried orpartially-dried hydrogel with the water-based pie-treatment agent. Theremineralisation gels or pastes (7) may be based on organic hydrogels orpastes. The hydrogel should be non-toxic, non-irritant and easilymouldable to the tooth contour. Examples of such hydrogels are thenon-reactive hydrogels mentioned above. These viscous gels would haveviscosities on the order of 100,000 to 1,000,000 cp. Solutions orpreparations with lower viscosities, such as aqueous solutions andglycerin-based compositions can also be used. Generally, neutral pH gelsare advantageous; however, the pH is preferably optimized to allow theionized form of the pre-treatment or remineralization agent to exist ata sufficient concentration.

The Tooth-Whitening (TW) and pre-treatment procedure is implemented witha similar kit, comprising of the above parts with the addition of:various tooth whitening agents in the form of a gel, paste or liquidsubstituted for, or used in addition to, the remineralisation agent (7).In addition, the EAER pen supplied would be modified with the TW (ToothWhitening voltage modulation programme memory card and/or processor. Thegels or pastes would be organic-based as outlined above.

Throughout the description and claims of this specification, thesingular encompasses the plural unless the context otherwise requires.In particular, where the indefinite article is used, the specificationis to be understood as contemplating plurality as well as singularity,unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties orgroups described in con_(j)unction with a particular aspect, embodimentor example of the invention are to be understood to be applicable to anyother aspect, embodiment or example described herein unless incompatibletherewith.

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of the words, for example“comprising” and “comprises”, mean “including but not limited to”, andare not intended to (and do not) exclude other moieties, additives,components, integers or steps.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described by way of examples only and withreference to the accompanying figures:

FIG. 1 is a graph showing the effect of pre-conditioning oniontophoretic treatment of a tooth. This figure shows current-timeresponses of a tooth obtained using a saline pad to complete the circuitbetween the working and reference of the counter-electrodes;

FIG. 2 shows a comparison of two lesions in one tooth before and aftertreatment using CPP-AVP as the remineralising agent; and

FIG. 3 shows (a) an incisor tooth before any treatment, (b) afterpre-conditioning and (c) after the iontophoresis-remineralising methodhas been applied.

EXAMPLES Example 1

In this experiment the current-time responses of an extracted toothafter the application of -I V at the working electrode were recorded.One electrode the shorted reference/counter electrode) was a 0.5 mmstainless steel wire inserted into the tooth root. The other electrode(the working electrode) was a Pt sheet electrode of area ca 0.25 cm²held in contact with a saline-soaked tissue pad, which in turn was heldin contact with the tooth surface close to the enamel lesion.

FIG. 1 shows the saline response (upper dotted line) measured after thetooth was previously held in contact with a hypochlorite-soaked pad for3 mins. The initial current after this topical hypochloritepre-treatment was 18 μA, over 20% higher than that of the tooth beforepre treatment, and the extended-time current was similar. The lower,solid trace shows the saline tooth response for the same tooth measuredafter being held in contact with a hypochlorite pad underelectrically-assisted (EA) pre-treatment for 3 mins at −1 V applied atthe working electrode. The initial current is similar, but theextended-time current is over five times larger (i.e. the current ismore negative, being lower down the negative current scale) after EAhypochlorite pre-treatment.

Example 2

FIG. 2 shows a comparison of two lesions in one tooth before and aftertreatment using CPP-ACP (Tooth Mousse) as the remineralising agent.Analysis of the mean mineral density of the lesions resulted inDemineralisation Parameters of 0.76 (left side) and 0.83 (right side)prior to treatment and 0.92 (left) and 0.83 (right) after treatment.This Demineralisation Parameter was derived by as comparison of averagegrey-scale levels within the Micro-CT image of: a) the lesion and b) thehealthy tissue.

This in vitro demonstration indicates that, applying a current at alevel safe and not perceived by patients at a fixed voltage to apre-conditioned natural caries lesion, in combination with CPP-ACP inthe form of Tooth Mousse resulted in significant (approximately 67%)remineralisation of the lesion (as measured by Image Analysis ofMicro-CT images of the tooth before and after treatment) after 3 hourselectrophoresis/iontophoresis application. The passive application ofthe agent Tooth Mousse-Plus (also known as MI paste) to the othernatural caries lesion on the same tooth for 3 hours resulted in minimalremineralisation (measured on Micro-CT images).

The comparison in FIG. 2 is of two lesions in one tooth before and aftertreatment

The images represent an approximately 10 micron thickness horizontalMicro-CT

(XCT slice) through the same path of the tooth with separate mesial anddistal lesions. The XCT image on the left shows the lesions prior to anytreatment. The image on the right shows the lesions after the lesionswere pre-treated to remove protein and lipids. The lesion on the leftwas treated with CPP-ACP and iontophoresis for three hours, whilst thelesion on the right was treated only with CPP-ACP plus Fluoride

(MI paste) for three hours.

Example 3

FIG. 3 shows an incisor tooth before any treatment, afterpre-conditioning and after the iontophoresis-remineralising method hasbeen applied.

The uppermost image shows an extracted incisor tooth which exhibits botha large carious cavity (caused by tooth decay), which is significantlydiscoloured, and areas of dark discolouration on the labial (fiat)facing surface of the crown of the tooth, adjacent to the canons cavityin the direction of the incisal (lower) edge of the tooth. This imagewas taken prior to any treatment being carried out.

The middle image shows the same tooth after 2 minutes ofpre-conditioning with sodium hypochlorite solution. There is very littledifference between the uppermost and middle images in terms of toothdiscolouration.

The lowermost image shows the tooth after theiontophoresis-remineralisation has been carried out using Tooth Mousse(CPP-ACP) as the re-mineralising agent for 1 hour. It is clear that thecavity has now lost its dark discolouration completely. The darkdiscolourations in the enamel of the crown of the tooth adjacent to thecavity have also disappeared. There is some increased whitening of theedges of the carious cavity at both the upper and lower margins of thecavity.

These images demonstrate the tooth-whitening effect of theiontophoresis-remineralising method.

What is claimed is:
 1. A method of remineralising a tissue comprising:providing a remineralising agent including a first component and asecond component: providing a first electrode having a first polarityand a second electrode having a second polarity, or providing the firstelectrode having the first polarity for a predetermined period of timeand subsequently reversing the first polarity to the second polarity;driving the first component of the remineralising agent into the tissueby applying iontophoresis with the first polarity and driving the secondcomponent of the remineralising agent into the tissue by applying theiontophoresis with the second polarity; and depositing theremineralising agent within the tissue,
 2. The method according to claim1, further comprising: pre-conditioning the tissue to remove proteinand/or lipids and then applying the remineralising agent whilstseparately, sequentially or simultaneously applying the iontophoresis.3. The method according to claim 1, further comprising: remineralisationof a hypo-mineralised or a de-mineralised tooth.
 4. The method accordingto claim 3, further comprising: performing a cosmetic treatment of thetooth.
 5. The method according to claim 4, further comprising:performing tooth lightening or whitening.
 6. The method according toclaim 1, further comprising: performing a preventive treatment of tootherosion.
 7. The method according to claim 1, further comprising:remineralising of a bone.
 8. The method according to claim 1, whereinthe remineralising agent comprises casein phosphopeptide-amorphouscalcium phosphate (CPP-ACP).
 9. The method according to claim 1, whereinthe remineralising agent is a fluoride-containing remineralising agent.10. The method according to claim 9, wherein the fluoride-containingagent is casein phosphopeptide-amorphous calcium fluoride phosphate(CPP-ACFP).
 11. The method according to claim 1, wherein theremineralising agent includes one or more remineralisation enhancers.12. The method according to claim 1, wherein the first component is asource of calcium ions and the second component is a source of phosphateions.
 13. The method according to claim 11, wherein the remineralisationenhancer is strontium
 14. The method according to claim 2, wherein thepre-conditioning is performed, with or without the application ofiontophoresis, prior to application of the re mineralising agent. 15.The method according to claim 1, wherein the pre-conditioning, comprisestreatment with an acid.
 16. The method according to claim 15, whereinthe acid is phosphoric acid.
 17. The method according to claim 2,wherein the pre-conditioning comprises treatment with a hypochlorite.18. The method according to claim 1, wherein the method furthercomprises the treatment or alleviation of dental caries and/or dentalfluorosis in a mammal.
 19. The method according to claim 1, wherein themethod further comprises the remineralising of hypo-mineralised orde-mineralised (carious) dentine.
 20. The method according to claim 1,further comprising: remineralising of a tooth of a mammal, wherein acounterelectrode is provided within a mouth of the mammal.
 21. Themethod according to claim 20, remineralising a lesion of the tooth. 22.The method according to claim 1, further comprising: depositing theremineralising agent as an orthophosphate within a dentine of the tooth.23. The method according to claim 1, further comprising: driving atleast one of the first component or the second component of theremineralising agent into the tissue by applying iontophoresis at aconstant voltage.